CN105018442A - Improved EN-apyrase - Google Patents

Abstract

The invention discloses improved EN-apyrase. The improved EN-apyrase has more complex glycosylation and longer biological half life in comparison with EN-apyrase disclosed by the prior art.

Description

The EN-apyrase improved

Technical field

The present invention relates to the improved form that name is called the prior art apyrase of EN-apyrase, called after I-EN-apyrase in this article.Therefore, in pharmaceutical field, the disease of that it is used for the treatment of thrombolysis (thrombolytic)-relevant or inflammation-relevant.

Background technology

PCT announces the EN-apyrase that WO2011/088244 describes the specific amino acid sequence with homology N-end, EN-apyrase molecule wherein more than 80% and homologue have the identical N-end started with ELVP, and have the sequence identical with hereinafter listed SEQ ID NO:180% or relative to the sequence of SEQ ID NO:1 only containing 1-5 preservative replacement.All EN-apyrases keep ADPase and ATPase active.Use specific construct in Chinese hamster ovary celI, obtain these EN-apyrases, described construct causes N-to hold homogeneity and has the transformation period of the raising being better than the apyrase being derived from soluble form CD39L3 in prior art.The characteristic of the application by regulating culture condition to improve this EN-apyrase, protein has been obtained under described culture condition, make to obtain even longer biological half time, supposition is because more complicated causes with higher levels of glycosylation.

Summary of the invention

Prepared one group of EN-apyrase improved, called after I-EN-apyrase, it has the pharmacokinetic properties being even better than EN-apyrase.

Therefore, in an aspect, the present invention relates to a kind of method improving the biological property of EN-apyrase, the method comprises and postpones glutamine to the interpolation in the substratum of Chinese hamster ovary celI until cultivate after one day, and described Chinese hamster ovary celI is modified with the expression construct containing described EN-apyrase.

In one aspect of the method, the present invention relates to a kind of EN-apyrase (I-EN-apyrase) of improvement, it has the transformation period of at least 23 hours in dog; And/or it has the molecular weight of at least 70kD; And/or its glycosylation pattern has the seminose 5+ seminose 8+G2FS2/ seminose 6+ seminose 7+G2FS1 ratio of raising.

accompanying drawing is sketched

Fig. 1 is the schematic diagram of the carrier for the preparation of EN-apyrase and I-EN-apyrase.

Fig. 2 shows under the culture condition of the protein for this improvement, stably produces the EN-apyrase of improvement in the going down to posterity for 42 times of specific cloning (350).

Fig. 3 shows compared with the more high molecular of EN-apyrase (being labeled as condition D) of the present invention, the difference of the molecular weight of the EN-apyrase (being labeled as condition A) of prior art, by measuring with the polygonal light-scattering analysis of HPLC-size exclusion chromatography coupling.

After Fig. 4 shows the EN-apyrase de-glycosylation of these two kinds of forms, polygonal light-scattering analysis that the is similar and coupling of HPLC-size exclusion chromatography.As shown in the figure, the molecular weight after de-glycosylation is identical.

Fig. 5 shows compared with the apyrase of prior art (condition A), the glycosylation pattern of EN-apyrase (condition D) of the present invention.

Fig. 6 compares the biological half time of the EN-apyrase (condition A) of the biological half time of I-EN-apyrase (condition D) of the present invention in dog and prior art.As seen, the transformation period of apyrase of the present invention in dog is much longer, and is approximately 25 hours.

embodiments of the present invention

Applicant finds the culture condition by regulating the EN-apyrase cultivating prior art, can obtain significantly improving of biological half time.It is believed that these glycosylation level of complexity owing to the level of glycosylation strengthened under these conditions and Geng Gao.Therefore, although there are some common features with the EN-apyrase of prior art, namely, EN-apyrase molecule wherein more than 80% and homologue have the identical N-end started with ELVP, and have identical with hereinafter listed SEQ ID NO:180% or relative to the sequence of SEQ ID NO:1 only containing 1-5 preservative replacement, but I-EN-apyrase of the present invention has the transformation period of enhancing and is characterised in that the glycosylation pattern of higher following ratio:

Seminose 5+ seminose 8+G2FS2/ seminose 6+ seminose 7+G2FS1.

Particularly, as shown in the following example, postpone to add in glutamine to substratum until first day causes the remarkable improvement of result after cultivating.

I-EN-apyrase of the present invention can be similar with the EN-apyrase to prior art mode be used for the treatment of the illness relevant with thrombosis with inflammation.Although describe these purposes in prior art in detail, in order to the convenience of reader, be summarized as follows:

purposes

In general at least apyrase-more specifically for any one in CD39 compound and CD39L1-8 as CD39L3 compound-purposes, I-EN-apyrase is useful therapeutical agent.These can be used as anti-platelet agents, the agent of resist dissolution thrombus, and can be used as anti-inflammatory agent and endotheliocyte (EC) protected protein.In addition, for treatment hemorrhagic conditions, EN-apyrase is useful in treatment, as U.S.8, and 535, described in 662, it is included in full by quoting.The illness can treated valuably by I-EN-apyrase comprises damage because machinery causes or drugs damage and causes hemorrhage illness.

Some clinical condition may require the release of the slow and prolongation of I-EN-apyrase or biological derivative.These situations may require I-EN-apyrase to be isolated in such as hydrogel or other pharmaceutically useful polymerizable gels.In addition, polyoxyethylene glycol (PEG) can be added and extend blood halflife, thus improve effect.When I-EN-apyrase is used as preventive medicine, single bolus dose administration can be used so that provide protection is maintained the longer time.Change that other of protein half-life are protein modified to be comprised, such as, albumin puted together, the change of IgG fusion molecule and Protein Glycosylation Overview pattern.

I-EN-apyrase can be used in ATP and/or ADP and be hydrolyzed into AMP and have in any clinical condition of clinical benefit, comprises the morbid state that wherein ATP and/or ADP concentration abnormality is high.Useful in the clinical condition that I-EN-apyrase plays an important role to the thrombocyte of wherein thrombocyte or activation in disease process such as metastases.

Clinical and the biological efficacy given interval after application of the I-EN-apyrase used is easily evaluated.Such as, when platelet count remains unchanged, use the bleeding time that cause more growing wherein.In addition, directly the measurement I-EN-apyrase of blood sample or the enzymic activity of metabolite also indicate the existence of this molecule in blood circulation.Based in conjunction with the method for the biochemical function for assessment of I-EN-apyrase known in the art, the transformation period of this albumen being estimated to the accurate sampling of blood sample.It will also be appreciated that other the clinical relevant tests for the existence of biological activity I-EN-apyrase.

the in vitro and in vivo appraisal procedure of instant apyrase effect

The biochemical function of I-EN-apyrase can by the available multiple method assessment of those of ordinary skill in the art.Such as, the enzymic activity of ATP enzyme and ADP enzyme can to contain in the solution of following material at 1ml in 37 DEG C and determines: 8mM CaCl ₂, 200 μMs of substrate (for ATP enzyme, substrate is ATP, or for ADP enzyme, substrate is ADP), 50mM imidazoles and 50mM Tris, pH7.5 (Picher etc., Biochem.Pharmacol. (1988) 51:1453).This reaction can be stopped and discharged inorganic phosphate (Baykov etc., Anal.Biochem. (1988) 171:266) can be measured by the Malachite Green Reagent adding 0.25mL.Based on the spectrophotometric analysis at 630nm place, the ATP enzyme (or ADP enzyme) of a unit corresponding to 1 micromole inorganic phosphoric acid salt at 37 DEG C/minute release.The crucial kinetic constant such as K of this enzyme _mand k _catcan obtain by fitting data in such as Michaelis-Menten equation.Other tests that can be used for monitoring bio chemical functional comprise, but be not limited to the radiometric analysis that described by (J.Clin Invest. (1998) 101:1851-1859) such as Gayle III and HPLC test, or the radiation-TLC described by Marcus, A.J. etc. (J.Clin Invest. (1991) 88:1690-1696) analyzes.

The biological function of EN-apyrase or derivative can by method assessment in ex vivo methods and body.The ex vivo methods that can be used for the biological function monitoring EN-apyrase and derivative comprises, such as, and platelet aggregation test (J.Clin Invest. (2002) 109:1031-1040 such as Pinsky, D.J.; Ozaki, Y, Sysmex J.Int. (1998) 8:15-22).

In the body that can be used for the biological function assessing I-EN-apyrase, method comprises mouse apoplexy model, measure the bleeding time, infarct volume, blood flow, neurological deficit, intracerebral hemorrhage and mortality ratio (Pinsky, D.J. etc., above; Choudhri, T.F. etc., J.Exp.Med. (1999) 90:91-99); Mouse ischemia of lung/re-perfusion model (Fujita, T. etc., Nature Med. (2001) 7:598-604); The Reperfu-sion apoplexy model (Huang, J. etc., Stroke (2000) 31:3054-3063) of baboon; Cd39-/-mouse (Pinsky, D.J. etc., J.Clin Invest. (2002) 109:1031-1040) and Yorkshire-Hampshire pig PCI model (Maliszewski, C.R. etc., PCT WO00/23094 (2000)) and the PCI model (Herbertm of rabbit, J-M. etc., ThrombHaemost (1998) 80:512-518; Fishman, J. etc., Lab Invest (1975) 32:339-351; Sarembock etc., Circulation (1989) 80:1029-1040).Those skilled in the art may know that other the apyrase strengthened for assessment of ADP enzyme and derivative are as the method for the biological function of thrombus regulatory factor (thromboregulator).

the therapeutic composition of I-EN-apyrase of the present invention

The invention provides the composition comprising the I-EN-apyrase of biologic effective dose of pharmaceutically acceptable dosage.These compositions or clinically after symptom can be applied to patient before symptom, during symptom.After symptom, using of I-EN-apyrase can be, such as, carries out after apoplectic seizure between 0 to 48 hour.Use can by such as injecting-intramuscular or subcutaneous, undertaken by suction, continuous infusion, sustained release or other pharmaceutically acceptable technology.Some clinical condition may require to use as single effective dose, or daily, can continue the longest one week or reach one month or the longer time.

Using is pharmaceutically acceptable form containing physiologically acceptable carrier, vehicle or thinner.Described thinner and vehicle can comprise neutral buffered saline solution, antioxidant (such as, xitix), low molecular weight polypeptide (such as, ≤ 10 amino acid whose polypeptide), amino acid, carbohydrate (such as, glucose, dextrose, sucrose or dextran), sequestrant (as, EDTA), stablizer (e.g., gsh).In addition, cosubstrate (cosubsatrate) can be used when administration, such as, calcium (Ca ²⁺), to obtain maximum enzyme activity.Select this kind of carrier nontoxic to patient under recommended dose and concentration and thinner.

Jointly can also use other reagent of the benefit strengthening independent I-EN-apyrase synergistically.Such as, use other anti-platelet agents or antithrombotics (as acetylsalicylic acid, heparin or Bivalirudin) can have extra benefit, such as, improve Reperfu-sion, extended treatment time window, prevent from blocking again and prevent microvascular thrombosis from being formed.Use I-EN-apyrase can strengthen effect and reduce thrombolytics ( tNKase ^tM, vampire plasminogen activator, urokinase, streptokinase, staphylokinase and ancrod) effective dose.Operated fusion polypeptide between the apyrase and thrombolytic agent of such as ADP enhancing can provide desirable treatment plan for Acute Myocardial Infarction (AMI), percutaneous coronary intervention (PCI) and acute ischemic stroke (AIS).

Significantly different according to following factor to the dose requirements of I-EN-apyrase: age, race, body weight, height, sex, treatment duration, medication, disease serious program or other clinical variables.Effective dose can be determined by experienced physician or other experienced medical workers.

Quoted passage provided by the invention is included in herein by quoting the theme quoted with regard to it.

preparation A

for generation of the construct of enhancement type apyrase and the Chinese hamster ovary celI with its conversion

Based on sol-CD39L3R67G T69R, devise the apyrase construct of coding EN-apyrase.Show this I-EN-apyrase and encoding sequence thereof as SEQ ID NO:1 below.

Signal sequence is ox alpha-lactalbumin signal peptide.For SEQ ID NO:1 express carrier in upstream sequence be SEQ ID NO:2.

Downstream sequence has SEQ ID NO:3.

Entire construct for inserting is SEQ ID NO:4.

The following construct being shown as SEQ ID NO:4 is inserted in retrovirus vector, and is called as " APT " in gained carrier shown in FIG.

Show the position for DNA fragmentation being cloned into MfeI and the XhoI restriction site in host range plasmid.Front 19 codon coded signal peptides.In the DNA sequencing process of final construct, detect silent mutation (AAC, and nonanticipating AAT) at position 2879 place.This codon adds double underline.Final carrier is called as oCS-APT-WPRE (new ori).

Chinese hamster ovary (CHO) production clone is by obtaining with the above retrovirus vector two-wheeled transduction CHO parental cell line shown in Fig. 1.By the transducer cell colony called after sCHO-S/sC-ATP-R2X converged.After each transduction, the sample of the clone colony converged is carried out freezen protective.By cell density (about 0.5 or 0.75 viable cell/200 μ L substratum) extremely low for the sCHO-S/sC-ATP-R2X cell colony dilution converged, and be seeded in 96 hole titer plate to set up the cloned cell line coming from single cell.After cultivation 14 days, clone the generation by Victoria Green WPB test screen EN-apyrase for totally 560.24 orifice plates are expanded to from 96 orifice plates based on 24 in the best clone that EN-apyrase produces.Survive in amplification, and be frozen preservation for 20 in described 24 clones.

In three T175 flasks repeated, these 20 clones are screened with regard to productivity.Selected the first five best clone is clone 176,248,290,350 and 372.The sCHO-S/sC-ATP-R2X cloned cell line selected is tested to retrovirus (RCR), mycoplasma contamination and the biological load with replication, result is negative.

preparation B

the cell culture condition of protein yields is improved in 10L bio-reactor

background: preliminary study shows, substratum obtains the more high yield of EN-apyrase and better glycosylation in shaking flask.Therefore, this is used in 10L bio-reactor.

materials and methods: clone CHO-S-APT-R was cloned #350 in every 3-4 days during exponential growth and go down to posterity, for the large-scale production of 10L Braun bio-reactor.? (Lonza) in substratum by cell with about 300,000-400, the cell density of 000 cell/ml is seeded in 10L bio-reactor.Batch feeding for this research is HyClone ^tMpS307 (12% (w/v) solution), AGT CD CHO5X Feed MediumComplete (Invitrogen), AGT CD CHO5X Feed Medium Complete+12.5g/L semi-lactosi (Invitrogen), 45% glucose solution, 20% glucose/galactose solution, 200mM L-glutaminate, the 50X solution of L-asparagine (15g/L)/Serine (10g/L), the 50X solution of TYR (4g/L)/CYSTINE (2g/L).

condition A

D0:3g/L PS307+2mM glutamine

D1: glutamine is maintained 2mM; Glucose is maintained 5g/L

D2:3g/L PS307+ glucose (to 5g/L)+glutamine (to 2mM)

D3: glutamine is maintained 2mM; Glucose is maintained 5g/L

D4:3g/L R15.4/PS307 (1: 1)+glutamine (to 2mM)+glucose (to 5g/L)

D5: glutamine is maintained 2mM; Glucose is maintained 5g/L

D5: by temperature change to 34 DEG C

D6：12g/L R15.4

D6: glutamine is maintained 2mM; Glucose is maintained 4g/L

D7-D16: glutamine is maintained 2mM; Glucose is maintained 4g/L

During whole service, pH is 7.4

At the 17th day harvested cell, and there is when gathering in the crops the protein level of 51mg/l.Lactate levels height about 6g/l, and ammonium level rests on lower than 10mM.

preparation C

the purifying of EN-apyrase is carried out with the rate of recovery improved

Pass through 1.4ft ²the substratum of 60M02Dpeth Filter (Cuno, CT) results cultured cells as described in preparation B.With WFI water washing filter before using, and blow off volume recovery is maximized with pressurized air.Then by the substratum 0.2 μm of frit through clarification, and be collected in sterile bag.

For inactivation of virus, by the Milli-Q water dilution of load (load) (substratum of 100mL through clarifying, V19) with equal-volume (100mL).Add x-100 solution (20mL, 11%) (final 1%), and gained solution is hatched 30 minutes at ambient temperature.

anion-exchange chromatography.EN-the substratum through inactivation of virus (220mL) of apyrase is applied to the flow velocity of 5ml/min and uses 10mM Tris-HCl, 5mL ANX Sepharose FF (GE Healthcare) post of pH7.4 balance.Load (load) is applied to this post, and collects stream and wear liquid and add washings (260mL).With 10mM Tris, this protein of 230mM NaCl, pH7.4 wash-out collecting (50mL).By skipping washing step and adopting the direct wash-out of 230mMNaCl, the apyrase from anion-exchange chromatography does not significantly lose.Finally, with the residual protein on 1M NaCl eccysis pillar, and this eccysis liquid is also collected (30mL).The western blot analysis of 1M NaCl band is shown, in this fraction, almost apyrase do not detected.

buffer-exchanged and cation-exchange chromatography.The ANX230mM elution volume collected is carried out buffer-exchanged (about 3 volume) to 20mM Citrate trianion, in pH4.6 by 1L G25 post.Load (150mL) through buffer-exchanged is applied at 20mM Citrate trianion, on 5mL SP-Sepharose FF (GE Healthcare) post balanced in pH4.6, and collects stream and wear liquid and washings (170mL).Use 20mM Citrate trianion, pH4.8 carries out washing step, and collects washings (40mL).Extra achievement is that the pH by stream being worn liquid is down to 4.6 from 4.8 and eliminates low molecular weight impurities.Although some apyrase is present in pH4.8 eluate, total amount be less than that pH4.6 stream wears liquid 10%.Use 20mM Citrate trianion, pH5.1 carries out another washing step, and collects washings (40mL).Use 20mM Citrate trianion, the residual protein on pH6.0 eccysis pillar, and this eccysis liquid is also collected (40mL).

Use Omega3kDa2ml centrifugal thickener carry out 20 × concentrated after, 4-12%SDS-PAGE gel is analyzed its purity.

By this purification schemes, have collected nearly all heterogeneous glycosylated EN-apyrase by ANX chromatography and eliminate 120mM and/or 140mM NaCl washing step.Equally, by reducing by 0.2 pH unit in SP chromatography, the apyrase (>95%) of higher degree is additionally obtained.By this two-step purifying, the total yield of apyrase is higher than 90%.

embodiment 1

improve the optimization of glycosylation and sialylated cell culture condition

background: preparation C research shows to be maintained in the 10L bio-reactor of 7.4 at pH, substratum result in the good glycosylation of EN-apyrase.New condition (D) is run in three 10L bio-reactors.

materials and methods: clone CHO-S-APT-R was cloned #350 in every 3-4 days during exponential growth and go down to posterity, for carrying out large-scale production in 10L Braun bio-reactor.? (Lonza) in substratum by cell with about 300,000-400, the cell density of 000 cell/ml is seeded in three 10L bio-reactors.Below list feeding strategy, pH and bio-reactor setting point.Results are for about 50%.

condition D

D0：3g/L PS307

D1: glutamine is maintained 2mM; Glucose is maintained 5g/L

D2:3g/L PS307+ glucose (to 5g/L)+glutamine (to 2mM)

D3: glutamine is maintained 2mM; Glucose is maintained 5g/L

D4:3g/L R15.4/PS307 (1: 1)+glutamine (to 2mM)+glucose (to 5g/L)

D5: glutamine is maintained 2mM; Glucose is maintained 5g/L

D5: by temperature change to 34 DEG C

D6：12g/L R15.4

D6: glutamine is maintained 2mM; Glucose is maintained 4g/L

D7-D16: glutamine maintains 2mM; Glucose is maintained 4g/L

During whole service, pH is 7.4

result: as follows from the peak value of the survivaling cell peak density of condition D: container Isosorbide-5-Nitrae 8.5 × 10 ⁵cell/mL, container 2,45.2 × 10 ⁵cell/mL and container 3,44.1 × 10 ⁵cell/mL.

Be prepared the 2-post process of C so that the I-EN-apyrase of this improvement is purified to homogeneity, make the biological load of the I-EN-apyrase of purifying be <2.0CFU/mL (Pass), and the intracellular toxin determined in the material (formulated bulk) of preparation is 2.0<X<4.0EU/mL or 0.6<X<1.2EU/mg.As shown in Figure 2, produce go down to posterity at 42 times in be stable.

The molecular weight (Fig. 3) of the molecular weight (70.4kD) of the I-EN-apyrase under condition D higher than 67.6kD under the condition A preparing B is shown with the polygonal light-scattering analysis of HPLC-size exclusion chromatography coupling.After de-glycosylation, molecular weight becomes identical (Fig. 4).

Therefore glycosylation composition between the protein example produced under condition A and D is quantitatively different.Although the content of Man6, Man7, G2FS1 is suitable, the protein produced under condition D contains Man5, Man8, G2FS2 of more high-content, and obtains acid and more complicated glycan structures.Therefore, the ratio of seminose 5+ seminose 8+G2FS2/ seminose 6+ seminose 7+G2FS1 is higher.The EN-apyrase of prior art be shown in Fig. 5 the comparing of glycosylation pattern of I-EN-apyrase.

embodiment 2

the transformation period extended and the pharmacodynamics of improvement

Pharmacokinetic has been carried out, wherein the single bolus of intravenous injection EN-apyrase or I-EN-apyrase in dog.Detect the ADP enzymic activity of serum sample.After EN-apyrase under application conditions A 6 hours, the apyrase removing about 60-80% from circulation was active.On the contrary, in this time period, the I-EN-apyrase under condition D remains 100% activity (Fig. 6).As directed, I-EN-apyrase in dog the transformation period more than 23 hours.

Claims (4)

1. one kind is improved the method for the biological property of EN-apyrase, the method comprises and postpones glutamine to the interpolation in the substratum of Chinese hamster ovary celI until cultivate after one day, described Chinese hamster ovary celI is modified with the expression construct containing described EN-apyrase, and wherein said expression construct comprises SEQ ID NO:4.

2. the process of claim 1 wherein that the biological property of described improvement is selected from:

A level of glycosylation that () is higher;

B glycosylation that () is more complicated;

C biological half time that () is longer; With

(d) its combination.

3. the EN-apyrase (I-EN-apyrase) improved, described EN-apyrase molecule wherein more than 80% and homologue have the identical N-end started with ELVP, and have identical with SEQ ID NO:180% or relative to SEQ ID NO:1 only containing the sequence of 1-5 preservative replacement, wherein said I-EN-apyrase has the transformation period of at least 23 hours in dog; And/or

It has the molecular weight of at least 70kD; And/or

Its glycosylation pattern has the seminose 5+ seminose 8+G2FS2/ seminose 6+ seminose 7+G2FS1 ratio of raising.

4. the EN-apyrase of claim 3, it has the glycosylation pattern shown in Fig. 5.